Hydrophilic polymers, such as polyethylene glycol (PEG), have been used for modification of various substrates, such as polypeptides, drugs and liposomes, in order to reduce immunogenicity of the substrate and/or to improve its blood circulation lifetime (Zalipsky, S. Adv. Drug Del. Rev., 16:157 (1995)).
For example, parenterally administered proteins can be immunogenic and may have a short pharmacological half-life. Proteins can also be relatively water insoluble. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients. Conjugation of PEG to proteins has been described as an approach to overcoming these difficulties. Davis et al. in U.S. Pat. No. 4,179,337 disclose conjugating PEG to proteins such as enzymes and insulin to form PEG-protein conjugates having less immunogenicity yet which retain a substantial proportion of physiological activity. Veronese et al. (Applied Biochem. and Biotech, 11:141–152 (1985)) disclose activating polyethylene glycols with phenyl chloroformates to modify a ribonuclease and a superoxide dimutase. Katre et al., in U.S. Pat. Nos. 4,766,106 and 4,917,888, disclose solubilizing proteins by polymer conjugation. PEG and other polymers are conjugated to recombinant proteins to reduce immunogenicity and increase half-life. (Nitecki et al., U.S. Pat. No. 4,902,502; Enzon, Inc., PCT/US90/02133). Garman (U.S. Pat. No. 4,935,465) describes proteins modified with a water soluble polymer joined to the protein through a reversible linking group.
However, PEG-protein conjugates described to date suffer from several disadvantages. For example, modification of the protein with PEG often inactivates the protein so that the resulting conjugate has poor biological activity, particularly for proteins that bind to a receptor or act on a macromolecular substrate. One approach to overcoming this problem is to attach the PEG chains at specific sites in the protein, avoiding the binding site to leave it unhindered for interaction. Alternatively, in some cases, the protein binding site can be protected (Caliceti et al., J. Bioactive Compat. Polym., 9:251 (1994)). However, these remedies are often difficult to implement. Typically, PEG in a PEG-protein conjugate is stably linked to the protein so that the beneficial properties provided by PEG remain. This in turn sometimes results in accumulation/vacuolization of conjugates that have difficulty to biodegrade (Bendele, A. et al., Toxicol. Sci., 42:152 (1998)). Having a cleavable linkage between the polymer and protein would alleviate these shortcomings.
PEG has also been described for use in improving the blood circulation lifetime of liposomes (U.S. Pat. No. 5,103,556). Here, the PEG is covalently attached to the polar head group of a lipid in order to mask or shield the liposomes from being recognized and removed by the reticuloendothelial system. Liposomes having releasable PEG chains have also been described, where the PEG chain is released from the liposome upon exposure to a suitable stimulus, such as a change in pH (PCT/US97/18813) or thiolysis (Kirpotin, D. et al, FEBS Letters, 38:115 (1996)). However, release of the PEG chain from the liposome suffers from the drawback that the decomposition products are chemically modified and can have unpredictable, potentially negative effects in vivo.